Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

BACKGROUND: Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. RESULTS: Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were analysed using MOG-displaying PHA granules and FACS analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range. CONCLUSION: E. coli can be genetically engineered to produce PHA granules displaying correctly folded eukaryotic proteins and which can be applied as beads in FACS based diagnostics. Since PHA granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads

Determinants and sources of iron intakes of australian toddlers: Findings from the SMILE cohort study

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. The first two years of life is a period of rapid growth and development. During this time a lack of key nutrients, including iron, can have long-lasting effects on motor and cognitive performance. The purpose of this cross-sectional study was to determine intake and sources of iron in a cohort of 828 toddlers (mean age; 13.1 mo) participating in the Adelaide-based Study of Mothers’ and Infants’ Life Events affecting oral health (SMILE), and to identify determinants of iron intake. At approximately 12 months of age, 3 non-consecutive days of dietary intake data were collected using a 24-h recall and 2-days food record. The Multiple Source Method was used to combine data from the 24-h recall and each day of the food record to estimate usual iron intake and descriptive statistics were used to report sources of iron. Linear regression was used to identify associations between iron intake and non-dietary determinants (maternal age, education, country of birth, BMI, socioeconomic position, parity, toddler sex) and primary milk feeding method at 12 months. The mean intake of iron was 7.0 (95% CI 6.7–7.2) mg/day and 18.2% of children had usual intakes below the estimated average requirement of 4 mg/day. The main sources of iron included infant and toddler cereals and formulas. Milk feeding method and parity were significantly associated with iron intake. Toddlers with siblings and those who received breast milk as their primary milk feed had significantly lower iron intakes than only children and those who received formula, respectively. The Australian Infant Feeding Guidelines promote the importance of iron-iron-rich complementary foods such as meat and meat alternatives. However, low intakes of this food group suggest that parents do not recognize the importance of these foods or understand the specific foods that toddlers should be eating

Assessment of gastroesophageal reflux symptoms and sleep quality among women in the Nurses\u27 Health Study II

IMPORTANCE: Limited data exist on the association of gastroesophageal reflux (GER) symptoms with sleep quality. OBJECTIVE: To prospectively investigate the association between GER symptoms and sleep quality. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study included data from the Nurses\u27 Health Study II of female nurses in the US. Participants self-reported the frequency and duration of GER symptoms beginning June 2005, with updates every 4 years through June 2015. Follow-up was completed June 2019, and data were analyzed from November 15, 2022, to June 4, 2023. EXPOSURES: Frequency and duration of GER symptoms. MAIN OUTCOMES AND MEASURES: Poor sleep quality was assessed in 2017 through a modified Pittsburgh Sleep Quality Index, which included difficulty in falling asleep, restlessness of sleep, daytime sleepiness, sleep disturbance, and sleep duration. Relative risk (RR) for poor sleep quality and individual components of poor sleep quality was estimated according to the frequency and duration of GER symptoms. RESULTS: Among 48 536 women (median age, 59 years [range, 48-69 years]), 7929 (16.3%) developed poor sleep quality during 4 years of follow-up. Compared with those with GER symptoms less than once a month, the multivariable RR for poor sleep quality among women with GER symptoms more than once a week was 1.53 (95% CI, 1.45-1.62). Women who had GER symptoms once or more a week for more than 7 years had an RR of 1.36 (95% CI, 1.30-1.43) compared with women who had not had GER symptoms once or more a week. The frequency and duration of GER symptoms were significantly associated with each individual component of poor sleep quality; for example, the multivariable RRs for GER symptoms 2 or more times per week compared with no GER symptoms were 1.49 (95% CI, 1.39-1.58) for difficulty in falling asleep, 1.47 (95% CI, 1.39-1.56) for excessive daytime sleepiness, and 1.44 (95% CI, 1.36-1.53) for restlessness of sleep. CONCLUSIONS AND RELEVANCE: In this prospective cohort study of female nurses in the Nurses\u27 Health Study II, the frequency and duration of GER symptoms were associated with subsequent risk of poor sleep quality. The findings suggest that effective treatment of GER disease may be important not only for improvement of symptoms but also for the reduction of comorbidities associated with poor sleep quality

A Comparison by Milk Feeding Method of the Nutrient Intake of a Cohort of Australian Toddlers

Breastfeeding is recommended beyond 12 months of age, but little is known about the contribution of breastmilk and infant formula to the nutritional intake of toddlers as they transition to a family diet in the second year of life. This study is a cross-sectional analysis of data collected from a birth cohort study in Adelaide, Australia. Dietary intake data were collected when children were approximately 1 year of age by an interviewer-administered multi-pass 24 h recall and a mother-completed 2 days food diary. Children were categorized according to their milk feeding method, i.e., breastmilk, infant formula, combination or other, and their nutrient intakes compared with recommended nutrient reference values. Complete data were available for 832 children, of which 714 had plausible energy intakes. Breastmilk and formula made a substantial contribution to the nutrient intake of those toddlers, contributing 28% and 34% of total energy, and 16% and 26% of protein intake, respectively when not drunk in combination. In general, Australian toddlers transitioning to the family diet consumed nutritionally adequate diets, although almost one quarter of all children and half of breastfed children with plausible intakes had iron intakes below the estimated average requirement, placing them at risk of iron deficiency

Pairwise approach for analysis and reporting of child’s free sugars intake from a birth cohort study

Objectives: The prospective cohort design is an important research design, but a common challenge is missing data. The purpose of this study is to compare three approaches to managing missing data, the pairwise (n = 1386 children), the partial or modified pairwise (n = 1019) and the listwise (n = 546), to characterize the trajectories of children's free sugars intake (FSI) across early childhood. Methods: By applying the Group-based Trajectory Model Technique to three waves of data collected from a prospective cohort study of South Australian children, this study examined the three approaches in managing missing data to validate and discuss children's FSI trajectories. Results: Each approach identified three distinct trajectories of child's FSI from 1 to 5 years of age: (1) ‘low and fast increasing’, (2) ‘moderate and increasing’ and (3) ‘high and increasing’. The trajectory memberships were consistent across the three approaches, and were for the pairwise scenario (1) 15.1%, (2) 68.3% and (3) 16.6%; the partial or modified pairwise (1) 15.9%, (2) 64.1% and (3) 20.0%; and the listwise (1) 14.9%, (2) 64.9% and (3) 20.2% of children. Conclusions: Given the comparability of the findings across the analytical approaches and the samples' characteristics between baseline and across different data collection waves, it is recommended that the pairwise approach be used in future analyses to optimize the sample size and statistical power when examining the relationship between FSI in the first years of life and health outcome such as dental caries

Translation, cultural adaptation and field-testing of the Thinking Healthy Program for Vietnam

Background: Depression and anxiety are prevalent among women in low- and lower-middle income countries who are pregnant or have recently given birth. There is promising evidence that culturally-adapted, evidence-informed, perinatal psycho-educational programs implemented in local communities are effective in reducing mental health problems. The Thinking Healthy Program (THP) has proved effective in Pakistan. The aims were to adapt the THP for rural Vietnam; establish the program’s comprehensibility, acceptability and salience for universal use, and investigate whether administration to small groups of women might be of equivalent effectiveness to administration in home visits to individual women. Methods: The THP Handbook and Calendar were made available in English by the program developers and translated into Vietnamese. Cultural adaptation and field-testing were undertaken using WHO guidance. Field-testing of the four sessions of THP Module One was undertaken in weekly sessions with a small group in a rural commune and evaluated using baseline, process and endline surveys. Results: The adapted Vietnamese version of the Thinking Healthy Program (THP-V) was found to be understandable, meaningful and relevant to pregnant women, and commune health centre and Women’s Union representatives in a rural district. It was delivered effectively by trained local facilitators. Role-play, brainstorming and small-group discussions to find shared solutions to common problems were appraised as helpful learning opportunities. Conclusions: The THP-V is safe and comprehensible, acceptable and salient to pregnant women without mental health problems in rural Vietnam. Delivery in facilitated small groups provided valued opportunities for role-play rehearsal and shared problem solving. Local observers found the content and approach highly relevant to local needs and endorsed the approach as a mental health promotion strategy with potential for integration into local universal maternal and child health services. These preliminary data indicate that the impact of the THP-V should be tested in its complete form in a large scale trial

Protocol for a process evaluation of a cluster randomized controlled trial of the Learning Club intervention for women\u27s health, and infant\u27s health and development in rural Vietnam

Background: Learning Clubs is a multi-component intervention to address the eight common risk factors for women’s health, and infant’s health and development in resource-constrained settings. We are testing in a cluster randomized controlled trial in rural Vietnam whether this intervention improves cognitive development in children when they are aged two. There are few comprehensive process evaluations of complex interventions to optimise early childhood development. The aim is to conduct a planned process evaluation of the Learning Clubs intervention in Vietnam. Methods: The evaluation will be conducted alongside the Learning Clubs trial using both qualitative and quantitative methods. Four domains will be included in the evaluation: [1] Context – how contextual factors affect the implementation and outcomes; [2] Implementation – what aspects of the Learning Clubs intervention are actually delivered and how well the intervention is delivered; [3] Mechanism of impact – how the intervention produces changes in the primary and secondary outcomes; and [4] National integration – how the intervention can be scaled up for application nationally. Purposive sampling will be used to recruit project stakeholders from commune, provincial and national levels. Results of the process evaluation will be integrated with those of the outcome and economic evaluations to provide a comprehensive picture of the effectiveness of the Learning Clubs intervention for early childhood development in rural Vietnam. Discussion: Results of the evaluation will provide evidence about the implementation of the intervention and explanations for any differences in the outcomes between participants in intervention and control conditions. The evaluation will be integrated into each stage of the outcome assessments, but will be implemented by a bilingual team independent of the team implementing the intervention. It will therefore provide evidence which will not be influenced by or influence the intervention and will inform both generalisation to other settings and scalability in Vietnam

Factors Associated With the Appropriate Use of Ultra-Broad Spectrum Antibiotics, Meropenem, for Suspected Healthcare-Associated Pneumonia

ABSTRACT: Pneumonia is a common disease-causing hospitalization. When a healthcare-associated infection is suspected, antibiotics that provide coverage for multi-drug resistant (MDR) or extended-spectrum beta-lactamase (ESBL) bacteria are frequently prescribed. Limited data is available for guidance on using meropenem as a first-line empiric antimicrobial in hospitalized patients with risk factors for MDR/ESBL bacterial infections. This was a single-center, retrospective study designed and conducted to identify factors associated with positive cultures for MDR/ESBL pathogens in hospitalized patients with suspected healthcare-associated pneumonia.Of the 246 patients, 103 patients (41%) received meropenem. Among patients prescribed meropenem, MDR/ESBL pathogens were detected in only 20 patients (13%). Patients admitted from a skilled nursing facility/long-term acute care (SNF/LTAC) or with a history of a positive culture for MDR/ESBL pathogens were significantly associated with positive cultures of MDR/ESBL pathogens during the hospitalization (odds ratio [95% confidence intervals], 31.40 [5.20-189.6] in SNF/LTAC and 18.50 [2.98-115.1] in history of culture-positive MDR/ESBL pathogen). There was no significant difference in mortality between the 3 antibiotic groups.Admission from a SNF/LTAC or having a history of cultures positive for MDR/ESBL pathogens were significantly associated with a positive culture for MDR/ESBL pathogens during the subsequent admission. We did not detect significant association between meropenem use as a first-line drug and morbidity and mortality for patients admitted to the hospital with suspected healthcare-associated pneumonia, and further prospective studies with larger sample size are needed to confirm our findings

Early Purchaser Involvement in Open Innovation- the case of an advanced purchasing function triggering the absorption of external knowledge in the French automotive industry

International audienceThis paper investigates the mechanisms that trigger the absorption of external knowledge in an innovative French automotive firm. An ethnographic-inspired study conducted by an academic embedded within the Innovation Purchasing Department has enable us to present a rare and new function of Purchasing that plays an important role between potential new suppliers and Research and Development personnel